Influencia de la penicilina y de las hormonas sexuales 17 beta-estradiol y progesterona sobre la infección por Chlamydia abortus en líneas celulares de endometrio y trofoblasto de origen ovino

Resumo

Chlamydia abortus is an obligate intracellular pathogen with affinity for placental tissues that causes reproductive failure in sheep and goats worldwide. In non-pregnant sheep, the organism can establish a latent or sub-clinical infection until the subsequent gestation, when it reactivates, invades the placenta and induces abortion. The precise mechanisms that trigger the awakening of C. abortus are still unknown. Previous studies suggest that fluctuations in hormones that regulate reproductive cycles in sheep, especially estradiol (E2) and progesterone (P4), might be associated with the reactivation of C. abortus. However, this association is poorly understood. The objectives of this PhD thesis are therefore: (i) to assess the influence of penicillin, E2 and P4 on the growth of C. abortus in vitro; (ii) to analyse the morphology of chlamydial inclusions grown in different cell lines and exposed to E2, P4 or penicillin, and (iii) to examine changes in the transcriptional response of C. abortus cultured in different cell lines and exposed to E2, P4 or penicillin. Experiments were carried out using two ovine cell lines: the trophoblast cell line AH-1 and the endometrial cell line LE. Cells were exposed to E2, P4 or penicillin prior to experimental infection with C. abortus. To study the influence of hormones and penicillin on the growth and development of C. abortus, we first performed an indirect immunofluorescence technique to assess the number and shape of inclusions in the different cultures. Then, we used supernatants to carry out the isolation and counting of viable free elementary bodies. Finally we collected cell monolayers to determine the amount of chlamydial DNA by q-PCR. To assess the effect of hormones and penicillin on the ultrastructure of inclusions, cell pellets were collected at 72 hours post-infection (hpi) and examined by transmission electron microscopy (TEM). Chlamydial transcriptional changes were studied from infected cells harvested at 48 and 72 hpi. mRNA expression analysis of 16 genes related to the chlamydial developmental cycle was carried out by q-PCR. Overall, sex hormones E2 and P4 enhanced the infectivity of AH-1 and LE cells, while penicillin had the opposite effect. Penicillin-treated C. abortus cultures showed, for the first time in this species, characteristic features of persistent infection such as appearance of small inclusions containing enlarged aberrant reticular bodies, and a gene profile characterised by general up-regulation of genes related to stress response and a down-regulation of genes related to membrane protein synthesis and cell division, regardless of the cell line used. The genes omcA and omcB (membrane proteins), dnaK (stress response) and hctA (conversion of reticular bodies to elementary bodies) are proposed as markers of the penicillin-induced persistent state in vitro in Chlamydia abortus. In contrast, the supplementation with hormones produced different results depending on the cell line used, suggesting that the effects of hormones on the chlamydial developmental cycle are indirect, via host cells. In addition, Progesterone and, to a lesser degree, 17?-estradiol induced the morphology of aberrant bodies in LE cultures, although this fact did not associate with alterations in studied genes in Chlamydia abortus. Therefore, a common pattern of gene expression, valid for different systems of chlamydial persistence induction, cannot be established.